Introduction: Cardiac fibroblasts (cFbs) maintain the extracellular matrix and provide a scaffold for cardiomyocytes (CMs). Following injury, cFbs become activated to myofibroblasts (MyoFbs) to aid in wound healing and repair. In disease, myofibroblasts contribute to pathological remodelling and cardiac fibrosis. This study aimed to investigate the potential for reversal of chronically activated human cardiac fibroblasts in 2D culture. We also evaluated the effects of quiescent and activated human cardiac fibroblasts on cardiomyocyte electrical and contractile function.

Methods: Primary human cFbs and human induced pluripotent stem cell derived cFbs (hiPSC-cFbs) were assessed for activation following 2-4 weeks of culture on stiff culture plastic (E = ~ 3 GPa) and in the presence of 10 ng/ml TGF-β. Myofibroblast reversal was assessed using TGF-β receptor inhibition (10 μM SB431542). mRNA and protein expression of activation markers α-SMA, collagens 1 and 3 were investigated. α-SMA expression was also assessed using immunofluorescence staining. Quiescent or activated iPSC-cFbs were plated on top of iPSC-CMs for 5 days. Contraction was assessed using MUSCLEMOTION and electrical activity using optical mapping.

Results: Primary cFbs and hiPSC-cFbs activate to myofibroblasts following culture on stiff plastic. In the absence of any chemical stimuli. Long term culture on stiff plastic led to both cell types becoming unresponsive to TGF-β stimulation or TGF-β receptor inhibition. hiPSC-cFbs remained quiescent in the presence of SB431542, while still being able to activate into MyoFbs upon TGF-β exposure (evident through increased α-SMA and collagen 1). Co-culture for five days with either cFb type did not affect CM contraction or calcium transients. TGF-β-exposed CMs showed increased action potential duration 50, 70 and 90 compared to SB431542-treated CMs, but no significant changes occurred with quiescent or activated fibroblasts.

Conclusions: Human cFbs activate independently of any external stimulus during standard 2D culture. Chronically activated human cFbs have a limited potential for reversal to a quiescent phenotype. Activation status of hiPSC-cFbs does not significantly affect contraction or electrical handling of iPSC-CMs.